Plant Cell Reports

Plant Cell Reports (1993) 12:343-346

9 Springer-Verlag 1993

Microprojectile transformation of sugarcane meristems and regeneration of shoots expressing fl-Glucuronidase Rhonda L. Gambley 1, Rebecca Ford 1, 2, and Grant R. Smith 1 1 David North Plant Research Centre, Bureau of Sugar Experiment Stations, PO Box 86, Indooroopilly, Queensland 4068, Australia Department of Biochemistry, University of Queenstand, St Lucia, Queensland 4072, Australia

2 Current Address:

Received September 18, 1992/Revised version received January 19, 1993 - Communicated by J. K. Vasil

of embryogenic callus was reported (Bower and Birch 1992).

Summary Microprojectile bombardment was used to introduce the GUS reporter gene into sugarcane axillary meristems. Chimeric expression of this gene was observed in 2040% of shoots regenerated from sugarcane meristems one month after particle bombardment. The linear pattern of GUS expression observed is consistent with periclinal division from single transformed meristematic cells. Meristems have advantages over callus cells as targets for microprojectile transformation, and have potential for introducing agronomically important genes into current commercial sugarcane varieties. Keywords: sugarcane, meristem, transformation,/3-glucuronidase, GUS

microprojectile

Introduction Commercial sugarcane cultivars, complex polyploid hybrids of S a c c h a r u m L. species, are the predominant crop in many areas of coastal Queensland, Australia. Progress in breeding by conventional crossing programs is difficult, as backcrossing for the introduction of specific genes is complicated by random genetic rearrangements occurring at meiosis. Genetic engineering is an attractive potential solution for the introduction of specific genes into sugarcane without genetic rearrangement. Considerable effort has been expended to develop transformation systems for sugarcane, including electroporation of protoplasts (Chela et al. 1987; Chowdhury and Vasil, 1992; Rathus and Birch, 1992) and microprojectile bombardment of suspension cells (Franks and Birch, 1991; Chowdhury and Vasil, 1992). The regeneration of plantlets from sugarcane protoplasts is difficult with very limited success (Srinivasan and Vasil, 1986; Taylor et al. 1992). Recently, the production of transgenic sugarcane plants following microprojectile bombardment Correspondence to: G. R. Smith

Previous studies have reported that sugarcane plants regenerated from callus show more somaclonal variation than plants regenerated from meristems (Irvine et aL 1991). Regeneration of plants from meristems is also considerably quicker than regeneration from callus. Maize (Klein et al. 1988) and soybean (McCabe et al. 1988) meristems, and soybean embryonic axes (Christou 1990) have been successfully used as biolistic transformation targets. Embryos of pearl millet (Taylor and Vasil, 1991) and germinating wheat (Lonsdale et al. 1990) have also been used as targets for transformation. While a microprojectile transformation system has been demonstrated for sugarcane (Bower and Birch 1992), the somaclonal variation evidenced in sugarcane plants regenerated from callus (Irvine et al. 1991) may preclude its use for practical sugarcane genetic engineering. Here, we report the development of a sugarcane meristem transformation system utilising the GUS (g-glucuronidase) reporter gene, as a preliminary to introducing and expressing genes of agronomic importance in transgenic sugarcane. Materials and methods

Plasmids and DNA preparation. Plasmid pEmuGN contains the Emu promoter, GUS (uidA) gene and Nos termination sequence in plasmid p U C I I 8 and was a kind gift from Dr D. Last (Last et aL 1991). Plasmid DNA was precipitated onto gold particles (1.5 to 3/~m) (Aldrich) at a concentration of 2/gg DNA per mg of gold using calcium chloride (2.5M) and spermidine (0.1M) at a ratio of 2.5:1, on ice. Final suspension concentration was 40mg of gold in 200#1. Nylon macro-projectiles as described (Franks and Birch, 1991) were loaded with 6/~1 of freshly vortexed suspension and kept on ice until firing. Planl material. Axillary buds were aseptically removed from the top metre of mature (6-9 months old) stalks of sugarcane varieties Q95 and Q137 and placed on medium containing Mnrashige and Skoog

344 (1962) inorganics, 3% (w/v) sucrose, 0.2 mg 1"1 kinetin and 0.1 mg 1q BAP (6-benzylaminopurine) and solidified with 3 g t 1 Gel-gro (ICN). These explants were cultured at 28~ under a 12h light/dark regime for 1 month prior to firing, After 1 month of culture, shoots were removed using a sterile scalpel to expose the meristematic tissues. The exposed tissue was placed on 0.8% (w/v) agar (Davis) in 65mm diameter polystyrene Petri dishes (Disposable Products) for use as a target.

Microprojectile Bombardment. The particle bombardment device as described by Franks and Birch (1991) used 'Ramset' charges (T19) for propulsion of the projectiles. The distance the macroprojectile was placed down the gun barrel was varied (4, 4.8, 5.5 or 6.8cm). After bombardment the meristems were placed onto fresh medium (as above) for regeneration of shoots. After 1 month individual regenerated shoots were assayed for GUS activity. GUS assays. Leaf segments 5ram long from regenerated shoots were placed in microtitre plates containing 100#1 of GUS assay buffer per well. The GUS assay buffer contained potassium ferricyanide (5nuM), potassium ferroeyanide (5raM), Triton X-100 (0.06% v/v) and 5bromo-4-chloro-3-indoyl-B-D-glucuronicacid (0.3% w/v). The plates were incubated in the dark for 2 days at 28~C, then the assay buffer removed and the wells filled with methanol to extract the chlorophyll from the leaf segments. The leaf segments were then examined under a dissecting microscope for GUS expression. Entire shoots were treated similarly and examined for GUS expression. Leaf segments from plantlets which had not been bombarded, or bombarded with microprojectiles without precipitated DNA, were used as negative controls. Plantlets from which sections were taken for analysis were placed in MS medium without added cytokinins for maintenance and root initiation.

p r o d u c e d p e r apex b y varieties Q95 and Q137 w e r e not statistically different (Table 1, t = 1.43, d f = 3 4 ) .

Expression o f GUS. G U S e x p r e s s i o n in excised leaf s e g m e n t s usually appeared as b l u e lines parallel to the m i d r i b o f the l a m i n a e (Fig. 1). C o n t r o l plantlets did not s h o w G U S activity. W h e n w h o l e plantlets w e r e placed in G U S substrate, b l u e p r o d u c t was o b s e r v e d t h r o u g h the centre o f the s h o o t base and as lines r u n n i n g up the l a m i n a e (Fig. 2). G U S activity was also o b s e r v e d in s o m e roots o f larger plantlets. T h e extent o f the G U S activity w i t h i n assayed plants appeared to be affected by the a b s o r p t i o n o f the G U S substrate. L a r g e plantlets s h o w e d G U S e x p r e s s i o n m a i n l y at the base (Fig. 2), w h e r e a s s m a l l e r plantlets appeared to express G U S m o r e e x t e n s i v e l y up the laminae.

DNA extraction. Leaf segments assayed as above were centrifuged at 17 000g for 15 minutes and the methanol discarded. The remaining tissue was ground in liquid nitrogen and 0.Sml of extraction buffer (IxSTE/0.1%SDS) and 0.5ml of phenol/chloroform (24:1) was added. This was centrifuged for 15min at 17 000g and the supernatant added to lml of cold absolute ethanol. After centrifuging for 30 minutes at 17000g the ethanol was discarded and the remaining pellet resuspended in 20#1 of TE buffer. PCR. Primers, GUS 1000 (5'-TTT GCA AGT GGT GAA TCC CGA CCT) and GUS 1600 (5'-AGT TTA CGC GTr GCT TCC GCC AGT) selected from the published sequence of the E. coli uidA gene (Jefferson et al. 1986) were synthesised on an Applied Biosystems PCR Mate 391 DNA synthesiser. A 50#1 PCR reaction mix contained these GUS-specific primers (10pmol each), AmpliTaq (2.5U), 2.5mM of each dNTP, lxPCR reaction buffer (Perkin Ehner) and 1#1 of extracted plant DNA. Reactions were overlaid with 50#1 of paraffin oil. PCR conditions used were 95~ for 5min followed by 30 cycles of 95~ 60~ 7TC/2min, with a 72~ final extension. Southern blotting. PCR products were separated in a 1% agarose gel and capillary blotted onto nylon membrane (N +, Amersham) using 100raM NaOH for 3h. Blots were then baked (80'C, 2h) and hybridised with pEmuGN linearised with EcoRl, and direct labelled with peroxidase (Amersham). ECL chemiluminescent detection was as per manufacturers' instructions (Amersham).

FIGURE 1. A sugarcane leaf segment showing GUS expression (12.8x). The leaf section was removed from a plantlet regenerated from a bombarded meristem one month after the bombardment. The dark lines running up the leaf blade, as indicated by the arrow, are deposits of the insoluble blue product resulting from GUS activity.

Table 1. Average number of shoots regenerated per sugarcane meristem one month after particle bombardment. Distance of the projectile down the gun barrel was 4.8cm. Sugarcane variety

Q137

Q95

21

15

Mean number of shoots regenerated

31.8

19

(SE)

(4.5)

(2.4)

Number of meristems bombarded

R~d~ Regeneration. L a r g e n u m b e r s o f shoots had regenerated f r o m the m e r i s t e m s o f varieties Q95 and Q137 one m o n t h after b o m b a r d m e n t (Table 1). T h e shoots varied in size but appeared healthy. T h e n u m b e r s o f shoots

Microprojectile bombardment. The distance the m a c r o p r o j e c t i l e was placed d o w n the barrel o f the particle gun affected the n u m b e r o f regenerated Q95 shoots w h i c h expressed the G U S g e n e (Table 2).

345 Fewer regenerated shoots showed GUS activity when the macroprojectile was placed 4cm down the barrel, than when it was placed at 4.8 or 5.5cm (X2=9.6, d f = l ; X2=9.5, d f = l ) . No significant difference was apparent between distances of 4.8 or 5.5cm (X2=0.01, df=l). For variety Q137, there was no significant difference in transformation frequency (Table 2, X 2= 1.3, d f = 1) between the assayed distances.

probe after Southern blotting, confirming it was part of the u i d A gene (Fig. 3). Negative control plant material was histochemically and PCR negative for the u i d A gene.

FIGURE 3. Southern blot of PCR products synthesised from DNA extracts of plantlets expressing the GUS gene. Lane I:PCR positive control (lng of pEmuGN); lanes 2 and 5: blank; lane 3:PCR negative control (no template); lane 4: No PCR product using genomic DNA from a control plant; lanes 6 to 13: 600bp product resulting from PCR using genomie DNA from plantlets expressing the GUS gene. Arrow indicatesposition of 600bp fragment. FIGURE 2. Glucuronidase expression in a plantlet regenerated from a meristem bombarded with a construct containing the GUS gene (8x). The dark areas at the base of the plantlet and the dark lines running up the leaves of the plant are the result of GUS expression. Table 2. Effect of distance macroprojectUe is placed down the gun barrel on the percentage of shoots per bombarded meristem expressing the GUS gene. Sugarcane variety Distance (em) Number of meristems bombarded Average number of shoots regenerated (SE) Average % of shoots transformed per meristem (SE)

Q95

Q 137

4.0

4.8

5.5

4.8

6.8

14

11

7

21

3

6

19

23

32

15

(1.6)

(2.4)

(4)

(4.5)

(5)

3

16

18

39

42

(1.5)

(5)

(8)

(6)

(19)

DNA extracts of those leaf segments which were histochemically GUS positive, yielded a 600bp fragment when primed with the specific primer pair in the PCR reaction. This fragment hybridised with the linearised

Discussion Expression of the fl-glucuronidase gene was observed in shoots regenerated from bombarded sugarcane meristematic tissue. The presence of the GUS gene was confirmed in histochemically positive tissue by PCR with primers specific for a 600bp region of the u i d A gene and Southern hybridisation of the PCR products with the plasrnid probe. Expression and confirmation of the presence of this reporter gene in regenerated tissue are important prerequisites to the production of transgenic sugarcane plants from bombarded meristems which express genes of agronomic importance. The pattern of gene expression observed in sugarcane plantlets was consistent with the blue streaks reported by Christou (1990) in plantlets derived from bombarded soybean embryonic axes. McCabe et al. (1988) also observed blue streaks in plants regenerated from bombarded soybean meristems. Christou (1990) suggested that the blue streaks he observed indicated a unicellular origin of the transformed cells comprising the blue streaks. Unlike the blue streaks observed by Christou (1990), those appearing in the sugarcane leaf segments usually appeared continuous, suggesting that the glucuronidase activity in daughter cells was the result of periclinal cell division from a single transformed meristematic cell.

346 GUS activity was not seen in control plantlets, and PCR on DNA extracted from this material did not synthesise any fragments which hybridised to the probe. While endophytic bacteria have been reported causing false positives in D i o s c o r e a transformation experiments with GUS as the reporter gene (Tor et al. 1992), we did not observe false positives with sugarcane, despite experimental controls to detect this phenomenon. Based on the pattern of GUS activity observed, the shoots derived from meristem transformation appear to be chimeric, although the blue colouration did predominate up the centre of small shoots and absorption of the GUS substrate appeared to be limited in large shoots. Meijer et al. (1991) previously reported prominent staining of vascular bundles near the cut end of rice leaves, suggesting that the degree of substrate uptake by different cell types appears to affect the observed GUS activity. The pattern of GUS activity, therefore, may not indicate the extent of transformation in a particular shoot. Chimeras may prove a problem for the successful expression of agronomically important genes, although the work of Irvine et al. (1991) on sectoring natural sugarcane chimeras into the constituent phenotypes, indicates that it may be possible to produce fully transgenic plants by meristem transformation followed by conventional micropropagation, rather than by callus-based tissue culture. Lonsdale et al. (1990) reported a large area of GUS expression on a root surface derived from a bombarded wheat embryo. They suggested that this may have resulted from the transformation of a root meristematic cell, which then gave rise to a transformed cell lineage. In the current study, shoots were derived directly from the bombarded meristem tissue and roots formed later by dedifferentiation at the base of the shoots after transfer to a cytokinin-free medium. As cells expressing GUS occurred both in the shoots and in the roots, it is likely that the original transformed cells of the sugarcane meristem dedifferentiated to form both shoots and roots rather than being predetermined as in the embryos used by Lonsdale et al. (1990). In the current study, the distance the nylon macroprojectile is placed down the barrel of the gun appears to affect the transformation frequency, with shorter distances (ie closer to the explosive charge) resulting in less regenerated shoots expressing GUS. The bombardment distance has been previously reported to affect transformation frequency (eg Taylor and Vasil, 1991), and like other workers we suggest that this is probably the result of the velocity of the microprojectiles causing physical damage to the plant tissue. Sugarcane meristems are an attractive and viable alternative target tissue to sugarcane callus for

microprojectile transformation. This approach contrasts that of Bower and Birch (1992) who used embryogenic callus and antibiotic selection to produce transgenie plants expressing the NPTII gene. Shoots regenerated from bombarded meristems show GUS activity at high frequencies (up to 40 %) in distance-optimised experiments. This is considerably higher than previously reported for GUS expression in bombarded sugarcane suspension cells or callus (Franks and Birch, 1991). A high frequency transformation system may be important for the introduction of agronomically important genes such as the SCMV coat protein gene (Smith et al. 1992), especially if a selectable marker, such as antibiotic resistance, can not be utilised for posttransformation selection. The other advantage of meristem transformation is the rapid regeneration of plantlets, with no apparent phenotypic changes, from target tissue. The use of meristems as transformation targets allows regeneration of large, vigorous shoots, a considerable advantage over the more frequently reported use of suspension ceils or callus as target tissue. Thus a gene of agronomic significance could be introduced into current sugarca)ae cultivars without altering other important agronomic traits. As a number of other varieties tested regenerate shoots readily after bombardment (unpublished data), this method should allow the transformation of a range of commercial sugarcane varieties. Acknowledgments: The authors thank Dr David Last for his kind gift of pEmuGN, and Mr Paul Taylor and Mrs Snezana Dukic for advice on sugarcane bud culture. We gratefully acknowledge the financial and logistical support of the Bureau of Sugar Experhnent Stations and the Sugar Research and Development Corporation. References Bower R, Birch RG (1992) The Plant J. 2(3):409-416 Chen WH, Gartland KMA, Davey MR, Sotak R, Gartland JS, Mulligan BJ, Power JD, Cocking EC (1987) Plant Cell Rep. 6:297-301 Christou P (1990) Ann. Bot. 66:379-86 Chowdhury MKU, Vasil IK (1992) Plant Cell Rep. 11:494-98 Franks T, Birch RG (1991) Aust. J. Plant Physiol. 18:471-80 Klein TM, Gradziel T, Frmmn ME, Sanford JC (1988) Biotech. 6:559-63 Irvine JE, Benda GTA, Legendre BL, Machado GR Jr (1991) Plant Cell Tissue Org. Cult. 26:115-25 Jefferson RA, Burgess SM, Hirsch D (1986) Proe. Natl. Aead. Sci. USA 83:8447-8451 Last DI, BretteU RIS, Chmnberlain DA, Chaudhury AM, Larkin PJ, Marsh EL, Peacock WJ, Dennis ES (1991) Theor. Appl. Genet. 81:581-8 Lonsdale D, Onde S, Craning A (1990) J.Exp.Bot. 41:1161-5 McCabe DE, Swain WF, Martinell BJ, Christou P, (1988) Biotech. 6:923-6 Meijer EGM, Schilperoort RA, Rueb S, van Os-Ruygrok PE, Hensgens LAM (1991) Plant Mol. Biol. 16:807-20 Mur~shige T, Sknog F (1962) Physiol. Plant. 15:473-97 Rathus C, Birch RG (1992) Plant Sci. 81:65-74 Smith GR, Ford R, Frenkel MJ, Shukla DD, Dale JL (1992)Arch.Virol. 125(14):15-23 Srinivasan C, Vasil IK (1986) J. Plant Physiol. 126:41-8 Taylor PWJ, Ko H-L, Adkins SW, Rathus C, Birch RG, (1992) Plant Cell Tissue Org. Cult. 28:69-78 Taylor MG, Vasil IK (1991) Plant Cell Rep. 10:120-5 Tor M, Mantell SH, Ainsworth C (1992). Plant Cell Rep. 11:452-56